The validity evaluation of different 16srRNA gene primers for helicobacter detection urgently requesting to design new specific primers

Molecular diagnosis of helicobacters by PCR is simpler, more accurate, and feasible compared to other diagnostic methods. Validity and accuracy are highly dependent on the PCR primer design, diffusion time, and mutation rate of helicobacters. This study aimed to design 16srRNA -specific primers for Helicobacter spp. and H. pylori. Application of comparative statistical analysis of the diagnostic utility of the most available 16srRNA genus-specific primers. The new primers were designed using bioinformatics tools (MAFFT MSA and Gblocks command line). A comparative study was applied on nine genus-specific 16srRNA primers in comparison to the ConsH using in silico and laboratory evaluation. The results demonstrated that the best specificity and sensitivity of the primers designed for this study compared to other primers. The comparative study revealed that the heminested outer/inner primers were the worst. Although H276, 16srRNA(a), HeliS/Heli-nest, and Hcom had acceptable diagnostic utility, false positive and false negative results were obtained. Specificity testing on clinical samples indicated a surprising result; that H. pylori was not the sole enemy that we were looking for, but the Non-Helicobacter pylori Helicobacters should be considered as a real risk prognostic for gastric diseases, consequently, a specific diagnosis and treatment should be developed. This study concluded that our designed primers were the most specific and sensitive in comparison with other primers. In addition, in silico evaluation is not accurate enough for primer assessment and that the laboratory evaluation is mandatory.


Results
The newly designed 16srRNA specific primers for Helicobacter spp. and H. pylori. The conserved regions of the 16srRNA gene-specific to all helicobacters and all H. pylori strains are demonstrated in Table 1; whereas the conserved regions used in the design of highly specific primers are demonstrated in Table 2. The first conserved area of Helicobacter spp. was used for designing the genus-specific primer (ConsH) and the second conserved area of H. pylori was used for designing the H. pylori specific nested primer (PyloA/ PyloAN). Comparative analysis between the newly designed primer ConsH and different 16srRNA primers for G: Helicobacter detection. In  Statistical evaluation of the diagnostic utility of different 16srRNA primers for Helicobacter spp. detection. ConsH matched all criteria considered for the gold standard selection. The statistical evaluation parameters according to in silico PCR amplification are described in Table 3. The outer/inner primers had 100% specificity and sensitivity with a P-value of 0.0001. Hcom1, H276, and HeliS/Heli-nest had 50% SP. The utility is measured also by the receiver operating characteristics (ROC) analysis expressed by the area under the curve (AUC), the ROC analysis of the in silico evaluation is demonstrated in Fig. 1. The laboratory evaluation showed 0.00% SP with heminested outer/inner primers with P-value=1. The H276, 16srRNA(a), HeliS/Heli-nest, and Hcom had  GTG GAT TAG TGG CGC ACG GGT GAG TAA CGC ATA GGT TAT GTG CCT CTT AGT TTG GGA TAG CCA TTG GAA  ACG GTG ATT AAT ACC AGA TAT TCC CTA CGG GGG AAA GAT TTA TCG CTA AGA GAT CAG CCT ATG CCC TAT  CAG CTT GTT GGT AAG GTA ATG GCT TAC CAA GGC TAT GAC GGG TAT CCG GTC TGA GAG GGT GAA CGG ACA  CAC TGG AAC TGA GAC ACG GTC CAG ACT CCT ACG GGA GGC AGC AGT AGG GAA TAT TGC TCA ATG GGG GAA  ACC CTG AAG CAG CAA CGC CGC GTG GAG GAT GAA GGT TTT AGG ATT GTA AAC TCC TTT TGT CAG AGA AGA  TAA TGA CGG TAT CTG ACG AAT AAG CAC CGG CTA ACT CCG TGC CAG CAG CCG CGG TAA TAC GGA GGG TGC  AAG CGT TAC TCG GAA TCA CTG GGC GTA AAG AGC GCG TAG GCG GGA TAG TCA GTC AGG TGT GAA ATC CTA  TGG CTT AAC CAT AGA ACT GCA TTT GAA ACT GCT ATT CTA GAG TGT GGG AGA GGC AGG TGG AAT TCT TGG  TGT AGG GGT AAA ATC CGT AGA GAT CAA GAG GAA TAC TCA TTG CGA AGG CGA CCT GCT AGA ACA TGA   www.nature.com/scientificreports/ 90% specificity with P-value= 0.001. Although 16srRNA(b) had 100% specificity, it had 75% sensitivity with a P-value=0.004. The ROC analysis of laboratory comparison is demonstrated in Fig. 2 and Table 4.

Discussion
The present study demonstrated the whole process of designing novel sets of highly specific primers targeting 16srRNA conserved region by bioinformatics tools, ConsH for detecting G: Helicobacter, and nested primers for H. pylori detection (PyloA/PyloAN). The design process was followed by evaluation of the diagnostic utility of the primers in comparison to a group of widely used 16srRNA primers in literature, and statistical monitoring of the diagnostic utility of the comparable primers used in Helicobacter spp. diagnosis. Efficient PCR performance is highly dependent on PCR primer design, consequently one must spend a notable effort on the primer design. Well-designed PCR primers not only augment specificity and sensitivity but also reduce the effort spent on the experimental optimization 42 .
PCR diagnosis especially nested PCR may be regarded as the gold standard for Helicobacter diagnosis through the construction of specific primers. Nested PCR provides higher sensitivity by excluding false-negative results due to low bacterial counts and PCR inhibitors 18 . PCR yielded a higher detection rate (40.8%) compared to histopathology (36.7%) and can be suitable for patients unfit for endoscopic examination 43 .
The choice of 16sRNA gene in PCR diagnosis of Helicobacter diseases was supported by a systematic review and meta-analysis conducted on various sources, including MEDLINE, Web of Sciences, and the Cochrane Library from April 1, 1999, to May 1, 2016. The most diagnostic candidate genes according to statistical parameters were 23S rRNA, 16S rRNA, and glmM 44 . The urgent demand for rapid accurate Helicobacter detection puts an obligation for the construction of novel specific primers.
Both the ConsH and PyloA/PyloAN primers were synthesized according to the bioinformatic tools with primer designs criteria which ensures efficient PCR performance. The primer' lengths fall within the recommended 18-30 nucleotides, shorter primers can produce non-specific results, and longer primers can form secondary structures and reduce PCR efficiency. Primers GC contents are within 48-52%, and the difference between the melting temperatures of the forward and reverse primers was within 4 °C versus other compared www.nature.com/scientificreports/ primers as 16srRNA (a), Hcom, and Heli-nest primers which have larger than 4 °C difference between the primers. This may cause false negatives 45,46 .
The evaluation of the newly designed primers was performed by in silico tools and laboratory test (PCR). The results of the in silico evaluation were very promising as ConsH matched with Helicobacters only and no mismatching with other bacteria, also the nested primers for H. pylori (PyloA/PyloAN) matches only with H. pylori. In silico PCR amplification showed highly encouraging results. The advantages of in silico evaluation are low cost and timesaving for evaluation as it gives a preliminary decision about the newly designed primer sets.  www.nature.com/scientificreports/ Nonetheless, the in silico evaluation is not a confirmatory method for evaluation due to continuously submitting uncurated sequences into GenBank, therefore, laboratory evaluation is mandatory 47 .
In the laboratory evaluation, the newly designed primers showed sufficient results. The specificity testing revealed significant results as ConsH detected all Helicobacters and the nested primer (PyloA/PyloAN) identified all five H. pylori strains without mismatching with Non-Helicobacter pylori Helicobacters (NHPH). They did not select any non-Helicobacter bacteria. The clinical precision testing by PCR was applied on gastric biopsies from dyspeptic patients introduced to the endoscope unit in the mentioned hospitals, revealing that all PCR results are consistent with the invasive methods applied (RUT and Histopathology). The positive PCR by sequencing confirmed the presence of Helicobacter DNA in the samples [48][49][50] The ConsH detected 40.7% positive samples, from the positive Helicobacter spp. PCR, the nested PCR detected 61.3% H. pylori and 39.7% Non-Helicobacter pylori Helicobacters (NHPH). These findings are concerning because there is an unexpected hidden unexpected enemy in the form of Non-Helicobacter pylori Helicobacters (NHPH), hence, diagnosis and treatment guidelines should be changed to consider the Non-Helicobacter pylori Helicobacter (NHPH) beside H. pylori.
In the present study, the designed primers showed considerable sensitivity to low concentrations of specific DNA in clinical samples, allowing sensitive detection in different types of contaminated samples 51 .
Here comes the answer to an important question about the need for a newly designed set of specific primers instead of the primers used in the literature. In silico and laboratory comparative analysis should be established to determine the diagnostic utility of 16srRNA primers for genus-level identification of Helicobacter spp. In silico comparison showed reasonable results for H276, 16srRNA(a), heminested outer/inner, and nested HeliS-Helinest by primer-blast but did not achieve substantial findings with 16srRNA(b). In silico PCR amplification gave promising results with H276 (96.8%), Hcom (98.4%), nonetheless, the results were poor for 16srRNA(b), BFHpyl, 16srRNA(a), and Heid primers. From the mentioned results, the in silico evaluation is not an accurate method for a real evaluation of any primer but is only a preliminary step, so laboratory evaluation is the confirmatory method for accurate evaluation. The specificity test revealed that ConsH primer is the best one in this marathon, as it has all considerations to be the gold standard primer for statistical evaluation of the comparable primers. The results of the laboratory concluded that the comparable primers offered false-positive results by non-specific binding to non-Helicobacter bacteria and some of them gave false-negative results as BFHpyl, Heid, and 16srRNA(b).
The comparative study was analyzed statistically to assess the diagnostic utility. The diagnostic parameters and ROC analysis of in silico and laboratory evaluation are demonstrated in Tables 3, 4, and Figs. 1, 2, which concluded that the in silico evaluation is not accurate enough to assess the diagnostic utility of any primer. That was evident in the case of heminested outer/inner primer which showed excellent diagnostic significance (P-0.0001), 100% in most diagnostic parameters, and the best score of AUC (1.00) in ROC analysis. These results differ in the laboratory evaluation. This heminested primer produced false-positive results because it achieved positive PCR with all non-Helicobacter bacteria with 0.00% specificity (Table 4) and AUC (0.5) (Fig. 2), which demonstrated the unreliability of heminested outer/inner primer for G: Helicobacter diagnosis. The aforementioned findings disagree with Qin et al. who confirmed its reliability for G: Helicobacter identification and that it is a powerful diagnostic tool 52 . Hcom1, H276, and nested primer (HeliS/Heli-nest) indicated non-significant results for their use in G: Helicobacter diagnosis by in silico PCR amplification (Table 3) and considered insignificant tool for diagnosis, as they had AUC less than 0.8 in ROC analysis tool. The laboratory evaluation of these primers demonstrated a major difference with in silico evaluation as the statistical analysis revealed that they had significant diagnostic utility (P-0.001) and were considered reliable for Helicobacter spp. diagnosis with AUC (0.950) in ROC analysis [53][54][55] . However, the results of the current study are incompatible with Flahou et al. that Hcom1 is suitable for the identification of Helicobacter spp. 56 , as it may give false-positive results with K. pneumonea. H276 may give positive results with S. mutans found in the buccal cavity 57 . The present study disagrees with Riley et al. who developed this primer and concluded that it was sensitive and specific to detect several numbers of Helicobacters and it was suitable for routine diagnosis 54 . The nested primers (HeliS/Heli-nest) gave positive results with Salmonella spp. which is alarming due to misdiagnosis of gastrointestinal disturbance of Helicobacteriosis and Salmonellosis 55 .
16srRNA (a, b) were unreliable based on in silico evaluation, but have a significance for diagnosis of Helicobacters (P-0.001 and 0.004, respectively) and an acceptable AUC (0.875, 0.950, respectively) in ROC analysis. However, false-positive results may be detected in 16srRNA (b) with E. fecalis. Low sensitivity obtained with 16srRNA(a) (Se 75%). Consequently, these results disagree with Idowu et al. and tiwari et al. who concluded their specificity and sensitivity in Helicobacter spp. diagnosis 55,58 . Finally, the worst primers that appeared in our study were clearly demonstrated in BFHpyl and Heid which have the lowest significance in in silico and laboratory evaluation (P-1.00) and low AUC (0.513, 0.450, respectively) by ROC analysis (Table 4) (Figs. 1, 2). These results disagree with Flahou et al. and Farshad 56,59 .
Our study explained a suitable comparative analysis of different primers for Helicobacters diagnosis by using ROC analysis and different statistical diagnostic parameters. Use of bioinformatics tools and command line to extract conserved regions of 16srRNA gene in Helicobacter spp. and H. pylori are used in the design of primers. Considerable detailed evaluation of the newly designed primers by specificity using sequenced Helicobacters and non-Helicobacters carefully selected from GIT microbes. Application of the designed primers in clinical samples (gastric biopsies) using sufficient representative sample size from dyspeptic patients introduced to three hospitals. The results were compared with the routine diagnosis in the hospitals and sequencing of the PCR products. Sensitivity testing was performed to find the detectable DNA concentration in highly contaminated samples (Stool). However, more non-Helicobacters and sequenced local Helicobacter strains should be used, and that will be considered in our future studies. www.nature.com/scientificreports/

Conclusion
Our designed primers ConsH and PyloA/PyloAN are highly sensitive and specific and could be used for accurate diagnosis of Helicobacter diseases from different clinical samples. Moreover, PyloA/PyloAN indicated the abundance of Non-Helicobacter pylori Helicobacter (NHPH) and their unrecognized role in Helicobacteriosis. It can also be concluded that in silico evaluation is not adequately accurate to assess the diagnostic utility of the primers and must be accompanied by the laboratory evaluation. Also, we recommend more research on Non-Helicobacter pylori Helicobacter (NHPH) and correlation with the clinical aspects of the disease in comparison with H. pylori.

Material and methods
Ethical statement. This study was approved by the Research Ethics Committee at Educational Hospitals and institutions committee (GOTHI) (Approval number; HAM00116). This committee confirms that all experiment was performed in accordance with relevant guidelines and regulations. Informed consent was assigned by all participants in the study (provided in supplementary files). Multiple sequence alignment (MSA). All 16srRNA gene sequences of all selected helicobacter strains were downloaded and saved in FASTA format to be used in MSA. MAFFT online version https:// mafft. cbrc. jp/ align ment/ server/ was used for MSA of all 16srRNA gene sequences of the selected strains with a distance matrix by counting the number of shared 6mers between every sequence pair. A guide tree was built, and the sequences were aligned progressively according to the branching order, then the tree was re-constructed, and finally, a second progressive alignment was carried out. MAFFT was applied twice to obtain the outputs of Pearson/FASTA and Clustal.  Comparative analysis between the newly designed primer ConsH and different 16srRNA primers for Helicobacter spp. detection. The 16srRNA specific primers used in the comparative study. The comparative study was performed on 9 primers for the detection of G: Helicobacter compared to the newly designed primer in this study (ConsH) ( Table 5).

Design new 16srRNA specific primers for G: Helicobacter and
In silico comparative study. These primers were examined by primer-blast and subjected to in silico PCR amplification.
Laboratory comparative study (specificity testing). PCR application of the comparable primers was performed on the sequenced Helicobacter strains and non-helicobacter strains.
Statistical evaluation of the diagnostic utility of different 16srRNA primers for G: Helicobacter detection in the literature. Gold standard primer selection. The selected gold standard primer for evaluation was the most specific one which gave negative results with all non-Helicobacter strains and positive results with all sequenced Helicobacter strains.